Cell culture assaysMouse epithelial

Cell culture assaysMouse epithelial LL/2 (LLC1) and mouse macrophages J774 cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) at 37 °C with 5% CO2. Adhesion experiments were performed as described by [8]. Bacterial adherence to the LL/2 cells was expressed as a percentage of the CFU of adhered bacteria compared tothe total number of CFUs of the initial inoculum. For the opsonophagocytic killing assay, mouse macrophages J774 were stimulated with 40 ng/ml E. coli LPS (Sigma-Aldrich) for 3 days. Macrophages (1 × 105 cells/ well) and A. baumannii strains (1 × 104 CFU/well) were added into the wells along with the heat-treated mice serum (56 °C for 40 min for the inactivation of complement components) at the final dilution of 1:200. After 1h incubation, the samples were serially diluted and seeded. Serum killing rates were counted by comparing the number of the reduced CFUs with those observed using naïve serum. A cytotoxicity assay was performed by incubating LL/2 cells with recombinant Blp1 C-terminal fragment in DMEM supplemented with 10% FBS at 37 °C for 24 h. Then, 20 μl of MTS (Promega) solution was added to the wells and cells were incubated for 4 h at 37 °C. After incubation, OD490 was determined. The proliferationrate was counted by comparing OD490 values for Blp1 C-terminus fragment-treated and non-treated cells.